PROJECT SUMMARY/ABSTRACT Musculoskeletal diseases are the most frequently reported medical conditions in the USA and the second- greatest cause of disability worldwide. Despite their medical importance, and the recognition that cellular topological relationships are key regulators in tissue development, we lack a fundamental understanding of how these relationships affect human skeletal development, maintenance, and repair, and how disruptions in topology lead to clinically relevant human disease. Prior studies showed that developing heterotopic ossification lesions had highly disorganized cellular structures. This contrasts sharply with the normal organized structures seen during skeletogenesis and suggests that changes in cell topology could contribute to disease pathogenesis. In this high risk/high yield proposal, we use new methods established by our team to elucidate how cellular topology affects bone development, and whether cellular topology contributes to the abnormal tissue integration that occurs in conditions of heterotopic ossification. We previously generated human induced pluripotent stem (iPS) cell lines from control subjects and subjects with fibrodysplasia ossificans progressiva (FOP), a congenital disease of massive heterotopic ossification induced by BMP signaling. We showed our ability to derive key bone cell lineages such as iPS cell-derived endothelial cells (iECs) and mesenchymal stem cells (iMSCs), and that the FOP iECs but not FOP iMSCs could show early activation of osteogenic fate. In Aim 1, we will use a new 3D bioprinting strategy to create ?bone organoids? with defined topological arrangements using our iMSCs and iECs from both control and FOP patients. This system will elucidate how ECs and MSCs interact during osteogenesis in different 3D configurations. In Aim 2, we will implant slices of these organoids into the skeletal muscle of SCID mice to test if cell arrangement contributes to the abnormal integration of bone into skeletal muscle as seen in heterotopic ossification. Our approach has several key innovations, including the use of FACS-defined human iPS cell- derived lineages which can be reproducibly isolated; and the use of a new scaffold-free 3D cell printing strategy that allows us to assess cell-cell interactions that may reflect normal bone development more closely, without complications introduced by a persistent structural matrix. Together, these studies will generate new fundamental information in a clinically relevant context for understanding how topologic arrangements influence osteogenesis by human cells. This new understanding may reveal the developmental mechanisms for potentiating osteogenesis, identify if topology helps regulate normal separation of bone and muscle, and identify novel targets for blocking the debilitating integration of muscle and bone that occurs in heterotopic ossification. Finally, these studies will support future efforts to create more complex multicellular structures using highly-defined cells to model human the complex regulatory process of human bone growth in vitro.